A bighorn sheep die-off in southern Colorado involving a Pasteurellaceae strain that may have originated from syntopic cattle

We investigated a pasteurellosis epizootic in free-ranging bighorn sheep (Ovis canadensis) wherein a Pasteurellaceae strain carried by syntopic cattle (Bos taurus) under severe winter conditions appeared to contribute to pneumonia in affected bighorns. Twenty-one moribund or dead bighorn sheep were found on the "Fossil Ridge" herd's winter range, Colorado, USA, between 13 December 2007 and 29 February 2008. Eight carcasses examined showed gross or microscopic evidence of acute to subacute fibrinous bronchopneumonia. All eight carcasses yielded at least one β-hemolytic Mannheimia haemolytica biogroup 1(±(G)) strain, and seven also yielded a β-hemolytic Bibersteinia trehalosi biogroup 4 (CDS) strain; evidence of Pasteurella multocida, Mycoplasma ovipneumoniae, and parainfluenza 3 and bovine respiratory syncytial viruses was also detected. Isolates of β-hemolytic Manneimia haemolytica biogroup 1(G) from a bighorn carcass and a syntopic cow showed 99.5% similarity in genetic fingerprints; B. trehalosi biogroup 4(CDS) isolates were ≥94.9% similar to an isolate from a nearby bighorn herd. Field and laboratory observations suggested that pneumonia in affected bighorns may have been caused by a combination of pathogens including two pathogenic Pasteurellaceae strains--one likely of cattle origin and one likely of bighorn origin--with infections in some cases perhaps exacerbated by other respiratory pathogens and severe weather conditions. Our and others' findings suggest that intimate interactions between wild sheep and cattle should be discouraged as part of a comprehensive approach to health management and conservation of North American wild sheep species.     

Synergistic Effects of Sodium Chloride,Glucose, and Temperature on Biofilm Formation by Listeria monocytogenes Serotype 1/2a and 4b Strains

Biofilm formation by Listeria monocytogenes is generally associated with its persistence in the food-processing environment. Serotype 1/2a strains make up more than 50% of the total isolates recovered from food and the environment, while serotype 4b strains are most often associated with major outbreaks of human listeriosis. Using a microplate assay with crystal violet staining, we examined biofilm formation by 18 strains of each serotype in tryptic soy broth with varying concentrations of glucose (from 0.25% to 10.0%, wt/vol), sodium chloride (from 0.5% to 7.0%, wt/vol) and ethanol (from 1% to 5.0%, vol/vol), and at different temperatures (22.5°C, 30°C, and 37°C). A synergistic effect on biofilm formation was observed for glucose, sodium chloride, and temperature. The serotype 1/2a strains generally formed higher-density biofilms than the 4b strains under most conditions tested. Interestingly, most serotype 4b strains had a higher growth rate than the 1/2a strains, suggesting that the growth rate may not be directly related to the capacity for biofilm formation. Crystal violet was found to stain both bacterial cells and biofilm matrix material. The enhancement in biofilm formation by environmental factors was apparently due to the production of extracellular polymeric substances instead of the accumulation of viable biofilm cells.Listeria monocytogenes, a Gram-positive bacterium, is capable of causing severe food-borne infections in both humans and animals. The organism is ubiquitous in the environment and can grow in a wide variety of foods, including those stored at refrigeration temperatures. It is particularly difficult to eliminate this bacterium from ready-to-eat foods and food-processing equipment (19). The ability to form biofilms protects the bacterium from stresses in food-processing environments (13, 25). Among the 13 different serotypes described, serotypes 1/2a, 1/2b, and 4b are involved in the majority of human cases of listeriosis. Serotype 4b strains have accounted for most human outbreaks, whereas the majority of L. monocytogenes strains isolated from foods or food-processing plants belong to serotype 1/2a (19).Comparative studies to link the phenotypic attributes of L. monocytogenes strains to serotypes have obtained variable results. Buncic et al. (4) have shown that serotype 1/2a isolates were more resistant to antilisterial bacteriocins than serotype 4b strains at 4°C. They also found that 4b isolates exhibited greater resistance to heat treatments at 60°C and were easier to recover than 1/2a strains immediately following cold storage. Bruhn et al. (3) observed that 1/2a strains (lineage II) grew faster than 4b and 1/2b (lineage I) strains in commonly used enrichment broth media (University of Vermont media I and II). However, other studies have indicated that similar differences could not be linked to a serotype (14), and sequencing results have shown a syntenic relationship between strains of the two serotypes (27).Some L. monocytogenes strains have consistently been isolated from food-processing plants over many years (1, 28). Although several studies have been carried out to identify differences in cell adherence and biofilm formation among different serotypes, conflicting results were obtained. Lineage I isolates (including serotypes 4b, 1/2b, 3c, and 3b) were found to produce higher-density biofilms than lineage II isolates (including serotypes 1/2a, 1/2c, and 3a) (8, 28). However, this conclusion was not supported by other studies (1, 7, 18). For serotype 4b strains, the capacity to form biofilms was reduced when the nutrient level in a medium decreased, while serotype 1/2a strains were not similarly affected (11).It has been suggested that the formation of a biofilm is a stress response by bacterial cells (15, 16). Biofilm research under laboratory conditions may not reflect biofilm formation in the environment. To investigate the behavior of L. monocytogenes in biofilms, a simulated food-processing (SFP) system including several stresses was designed (30). The SFP system was used to study 1/2a and 4b strains in mixed-culture biofilms (31). Bacterial cells from a 1/2a cocktail predominated over 4b strains when exposed to the SFP system for 4 weeks, but no competitive inhibition was observed. Environmental factors, including temperature, sugar, salt, pH, and nutrients that are common in foods and food-processing environments, have been demonstrated to have impacts on L. monocytogenes adhesion and biofilm formation (25). The objectives of this study were to investigate and compare biofilm formation between L. monocytogenes serotype 1/2a strains and serotype 4b strains under a variety of environmental conditions, including different temperatures and varying concentrations of salt, sugar, and ethanol, and to examine the synergistic effects of these factors on biofilm formation by both serotypes.     

Animal-to-Animal Variation in Fecal Microbial Diversity among Beef Cattle

The intestinal microbiota of beef cattle are important for animal health, food safety, and methane emissions. This full-length sequencing survey of 11,171 16S rRNA genes reveals animal-to-animal variation in communities that cannot be attributed to breed, gender, diet, age, or weather. Beef communities differ from those of dairy. Core bovine taxa are identified.The gastrointestinal tracts (GIT) of beef cattle are colonized by microorganisms that profoundly impact animal physiology, nutrition, health, and productivity (5). The GIT microbiota potentially impact food safety via pathogen shedding (13) by interacting with organisms such as Salmonella and competing for resources in the GIT. Cattle intestinal microbiota also play an important role in methane emissions, with U.S. beef cattle alone contributing an estimated 3.87 million metric tons of methane into the environment each year, both from rumen and large-intestine fermentations (7). Although the bovine fecal microbiota have been well characterized using culture-based methods, these techniques are necessarily limited to characterizing bacteria that can be grown in the laboratory. Culture-independent methods can reveal community members that are recalcitrant to culture. Only a handful of deep-sequencing studies have been done using culture-independent 16S rRNA-based methods (1, 11, 12, 14), all with dairy cattle, which have a fundamentally different diet and metabolism from beef cattle. Despite the potential contributions of the beef cattle GIT microbiota to animal health, food safety, and global warming, these communities remain poorly characterized. With the advent of pyrosequencing technology, researchers now have the tools to characterize these important communities. Pyrosequencing will allow rapid characterization of large-sample data sets (1). However, the taxonomic information generated by rapid sequencing is approximate by necessity (9), and full-length 16S-rRNA sequencing remains the “gold standard” method. Accordingly, we have characterized fecal bacteria from six feedlot cattle by full-length capillary sequence analysis of 11,171 16S rRNA gene clones (Fig. ​(Fig.11).Open in a separate windowFIG. 1.Bacterial diversity of six feedlot beef cattle. Gray bars represent the percentages of all 16S sequences that were assigned to each taxonomy. Colored dots represent the percentages of 16S sequences from each library that were assigned to each taxonomic group. Asterisks indicate unclassified members of the named taxon. Panel A shows the data for the first 99% of all the sequences. Panel B shows the data for the remaining 1% of sequences. Note differences in scales for panels A and B.Rectal grab fecal samples (n = 6) were collected according to institutional animal care guidelines. All animals were female cross-bred MARCIII beef heifers, 6 to 8 months of age, 214 to 241 kg, housed in the same feedlot pen for 2 months prior to fecal collection, and fed the same typical feedlot beef production growing rations consisting of 61.6% corn silage (41.3% dry matter), 15.2% alfalfa hay, 20.9% corn, and 2.3% liquid supplement.Total fecal DNA was isolated from homogenized samples using MoBio UltraClean fecal kit (Carlsbad, CA). PCR was performed using 27F and 1392R primers (11). Amplification consisted of 25 cycles, with an annealing temperature of 55°C. Amplicons from three reactions per sample were pooled (8), cloned using the Invitrogen TOPO TA cloning kit (Carlsbad, CA), and sequenced bidirectionally with M13 primers using an ABI 3700 sequencer (17). Low-quality and chimeric sequences (6) were excluded from further analysis. Distance matrices were compiled from ClustalW alignments (18) in PHYLIP (4). Pairwise estimates of shared richness were calculated using EstimateS, version 8.2 (R. K. Colwell; http://purl.oclc.org/estimates). DOTUR (16) was used to identify operational taxonomic units (OTUs) and to generate rarefaction curves (Fig. ​(Fig.2),2), richness and evenness estimates, and Shannon''s and Simpson''s diversity indices (Table ​(Table1).1). A 97% similarity cutoff and an 85% similarity cutoff for estimating OTUs were used to approximate species and class-level designations (15). Taxonomies were assigned to one member of each OTU using the RDP “classifier” tool (19), and the RDP taxonomic information was used for Fig. ​Fig.11 and ​and3.3. Common bovine taxa were identified based on inclusion in all three U.S. culture-independent studies (this study and references 1 and 11).Open in a separate windowFIG. 2.Rarefaction curves for six feedlot beef cattle. OTUs were assigned at the 85% DNA sequence similarity level. For comparison purposes, all six curves were truncated after 1,321 sequences.Open in a separate windowFIG. 3.Phylum-level distribution of bacterial sequences from six beef feedlot cattle. Asterisks indicate unclassified members of the named taxon.

TABLE 1.

Richness and diversity indices for 6 beef feedlot cattle

Heterotypic Humoral and Cellular Immune Responses following Norwalk Virus Infection

Norovirus immunity is poorly understood as the limited data available on protection after infection are often contradictory. In contrast to the more prominent GII noroviruses, GI norovirus infections are less frequent in outbreaks. The GI noroviruses display very complex patterns of heterotypic immune responses following infection, and many individuals are highly susceptible to reinfection. To study the immune responses and mechanisms of GI.1 persistence, we built structural models and recombinant virus-like particles (VLPs) of five GI strains: GI.1-1968, GI.1-2001, GI.2-1999, GI.3-1999, and GI.4-2000. Structural models of four GI genotype capsid P domain dimers suggested that intragenotype structural variation is limited, that the GI binding pocket is mostly preserved between genotypes, and that a conserved, surface-exposed epitope may allow for highly cross-reactive immune responses. GI VLPs bound to histo-blood group antigens (HBGAs) including fucose, Lewis, and A antigens. Volunteers infected with GI.1-1968 (n = 10) had significant increases between prechallenge and convalescent reactive IgG for all five GI VLPs measured by enzyme immunoassay. Potential cross-neutralization of GI VLPs was demonstrated by convalescent-phase serum cross-blockade of GI VLP-HBGA interaction. Although group responses were significant for all GI VLPs, each individual volunteer demonstrated a unique VLP blockade pattern. Further, peripheral blood mononuclear cells (PBMCs) were stimulated with each of the VLPs, and secretion of gamma interferon (IFN-γ) was measured. As seen with blockade responses, IFN-γ secretion responses differed by individual. Sixty percent responded to at least one GI VLP, with only two volunteers responding to GI.1 VLP. Importantly, four of five individuals with sufficient PBMCs for cross-reactivity studies responded more robustly to other GI VLPs. These data suggest that preexposure history and deceptive imprinting may complicate PBMC and B-cell immune responses in some GI.1-1968-challenged individuals and highlight a potential complication in the design of efficacious norovirus vaccines.Noroviruses are the second-most important cause of severe viral gastroenteritis in young children and cause approximately 20% of endemic familial diarrheal disease and traveler''s diarrhea in all ages (reviewed in references 45 and 70). Noroviruses are genetically grouped into five different genogroups (GI to GV). GI and GII genogroups are responsible for the majority of human infections and are subdivided into more than 25 different genotypes (for example, GI.1 is genogroup I genotype 1). Most norovirus outbreaks are caused by the GII.4 genotype (65). Although genogroup I strains are associated with fewer reported outbreaks, they are frequently identified in environmental samples and in children (7, 21, 33, 58, 74, 82). The severity of norovirus disease is usually moderate although infection can be especially virulent, even fatal, in the elderly (14, 24, 31, 38, 46, 67). An effective vaccine would be particularly advantageous to vulnerable older populations, food handlers, child and health care providers, and military personnel. One major obstacle to norovirus vaccine development is the lack of understanding of the extensive antigenic relationships between heterogenic norovirus family members and of how this antigenic heterogeneity affects host protective immunity. Norovirus heterogeneity can be examined through sequence, structural, ligand binding, and host immune studies.Structurally, noroviruses are ∼38-nm icosahedral viruses with an ∼7.5 kb single-stranded, positive-sense RNA genome that encodes three large open reading frames (ORFs). ORF1 encodes the replicase polyprotein, while ORFs 2 and 3 encode the major and minor capsid proteins, respectively. The ORF2 major capsid protein sequence can vary by up to 60% between genogroups and by ∼20 to 30% between the genotypes (91). Expression of the major capsid protein (ORF2) in baculovirus and Venezuelan equine encephalitis (VEE) results in formation of virus-like particles (VLPs) composed of 180 copies of the monomeric protein (72). The monomer is structurally divided into the shell domain (S) that forms the structural core of the particle and the protruding domain (P) that protrudes away from the core. The P domain is further subdivided into the P1 subdomain (residues 226 to 278 and 406 to 520) and the P2 subdomain (residues 279 to 405) (72). P2 represents the most exposed surface of the viral particle and determines interaction with both potential neutralizing antibody recognition sites and putative cellular receptors, the histo-blood group antigens (HBGAs) (13, 16, 54, 57).The P domain has been shown to independently form dimers and P particles comprised of 12 monomers (85). Dimers and P particles share structural and HBGA binding similarities with the VLP generated with the same monomers (9, 85, 87). Three norovirus-HBGA binding profiles have been identified: (i) those that bind A/B and/or H epitopes, (ii) those that bind Lewis and/or H epitopes, and (iii) those that do not bind any available HBGA (86). Elegant structural analyses of Norwalk virus VLPs in complex with synthetic HBGAs identified a highly conserved binding site within the G1 noroviruses and predicted that structural constraints within the GI strains would restrict HBGA binding patterns to either a terminal Gal-Fuc or GalNAc (18, 88).Norwalk virus (NV; GI.1-1968) is the prototypic GI strain and typically infects individuals who encode a functional FUT2 α-1,2-fucosyltransferase enzyme resulting in expression of HBGAs on mucosal surfaces (secretor-positive phenotype) (53). Individuals who do not encode a functional FUT2 enzyme have a secretor-negative phenotype, do not express ABH HBGAs on mucosal surfaces, and are resistant to NV infection. Outbreak investigations have confirmed the association between HBGA expression and norovirus infection for some GI and GII strains (37, 39, 43, 49, 89). It remains likely that enzymes other than FUT2 may function as norovirus susceptibility factors because secretor-negative individuals have low-level norovirus-reactive antibodies (49, 52, 53) and can become infected after challenge with a GII.2 strain (52); in addition, some norovirus strains bind to FUT2-independent HBGAs in vitro (35, 54, 79).Early challenge studies (reviewed in reference 50) suggested that short-term protective immunity may occur following NV challenge (96). Demonstration of long-term protective immunity has been more complex. One early rechallenge study found that 50% of NV-challenged volunteers experienced repeat infections after ∼3 years while the other 50% remained well initially and after repeated challenge (69). Whether these volunteers remained disease free because of acquired immunity or genetic resistance could not be ascertained (69). However, contemporary norovirus challenge studies suggest that an early mucosal IgA response is associated with protection from NV infection (53). Further, strong gamma interferon (IFN-γ) secretion from CD4⁺ T cells (52) was identified in some uninfected GII.2-1976-challenged volunteers.In the absence of additional rechallenge studies, the most compelling evidence for a long-term protective immune response comes from the growing number of reports from around the world indicating that periods of “high norovirus activity” correlated with the emergence of new GII.4 strains (1, 10, 42, 66, 75, 90). Subsequently, the years following the high activity were characterized by decreased numbers of outbreaks, indicating that herd immunity may be an important regulator of GII.4 noroviruses (54, 80, 81). Clearly, the molecular basis for differential protective immunity/susceptibility following repeat norovirus infection is complex and a major challenge for the field.In this report, we compare the VLP phenotypes of the prototypical norovirus strain NV to an extant GI.1 strain isolated 33 years after NV and to a panel of VLPs representing strains GI.2, GI.3, and GI.4. In the results, we evaluate sequence conservation, carbohydrate (CHO) binding patterns, and antigenic relatedness at the antibody and T-cell levels. In contrast to earlier predictions (19), these data suggest that the GI noroviruses can bind many different HBGAs and that individuals infected with norovirus usually mount robust B- and T-cell responses against homologous strains. Surprisingly, some individuals appear to preferentially mount immune responses against heterologous GI strains.     

A Novel Model of Lethal Hendra Virus Infection in African Green Monkeys and the Effectiveness of Ribavirin Treatment

The henipaviruses, Hendra virus (HeV) and Nipah virus (NiV), are emerging zoonotic paramyxoviruses that can cause severe and often lethal neurologic and/or respiratory disease in a wide variety of mammalian hosts, including humans. There are presently no licensed vaccines or treatment options approved for human or veterinarian use. Guinea pigs, hamsters, cats, and ferrets, have been evaluated as animal models of human HeV infection, but studies in nonhuman primates (NHP) have not been reported, and the development and approval of any vaccine or antiviral for human use will likely require efficacy studies in an NHP model. Here, we examined the pathogenesis of HeV in the African green monkey (AGM) following intratracheal inoculation. Exposure of AGMs to HeV produced a uniformly lethal infection, and the observed clinical signs and pathology were highly consistent with HeV-mediated disease seen in humans. Ribavirin has been used to treat patients infected with either HeV or NiV; however, its utility in improving outcome remains, at best, uncertain. We examined the antiviral effect of ribavirin in a cohort of nine AGMs before or after exposure to HeV. Ribavirin treatment delayed disease onset by 1 to 2 days, with no significant benefit for disease progression and outcome. Together our findings introduce a new disease model of acute HeV infection suitable for testing antiviral strategies and also demonstrate that, while ribavirin may have some antiviral activity against the henipaviruses, its use as an effective standalone therapy for HeV infection is questionable.Hendra virus (HeV) and Nipah virus (NiV) are members of the genus Henipavirus (family Paramyxoviridae) that can cause severe respiratory illness and/or encephalitis in a wide variety of mammals, including horses, pigs, and humans (7, 23). HeV was identified as the causative agent of an acute respiratory disease in horses in 1994 in Queensland, Australia (23), and to date there have been 14 outbreaks in Australia since, with at least one occurrence per year since 2006, most recently in May 2010 (ProMed-mail no. 20100522.1699 [International Society for Infectious Diseases, http://www.promedmail.org]). Every outbreak of HeV has involved horses as the initial infected host, and there have been a total of seven human cases arising from exposure to infected horses. Four human fatalities have occurred (22), with the most recent occurring in August of 2009 (ProMed-mail no. 20090826.2998 and 20090903.3098). All patients initially presented with influenza-like illnesses (ILIs) after an incubation period of 7 to 16 days. While two individuals recovered from ILI, one patient developed pneumonitis and died from multiorgan failure. Three of the lethal cases developed encephalitic manifestations (mild confusion and ataxia), with two patients experiencing seizures (22, 23, 27).Data on the histopathology of fatal human HeV cases are limited, but the pathology includes small necrotic plaques in the cerebrum and cerebellum, in addition to mild parenchymal inflammation (21, 27). Severe parenchymal inflammation and necrosis were observed in the lungs. More extensive histopathologic data are available from 32 autopsies of fatal human NiV cases (28). Similarly to the HeV cases, pathology was characterized by systemic vasculitis and parenchymal necrosis in the central nervous system (CNS), while in the lung, pathological findings mainly included vasculitis, fibrinoid necrosis, alveolar hemorrhage, pulmonary edema, and aspiration pneumonia. Other organs that were affected included heart, kidney, and spleen and showed generally mild or focal inflammation. The development of syncytial multinucleated endothelial cells is characteristic of both HeV and NiV (27, 28). At present, the details of the pathogenesis and histopathological changes mediated by either HeV or NiV infection in humans are naturally derived from only the late phases of the disease course, and therefore a relevant animal model is needed that mimics the disease progression seen in humans.Pteropid fruit bats, commonly known as flying foxes in the family Pteropodidae, are the principle natural reservoirs for both NiV and HeV (reviewed in reference 3). However, these henipaviruses display a broad species tropism, and in addition to bats, horses and humans, natural and/or experimental infection of HeV has been demonstrated in guinea pigs, hamsters, pigs, cats, and ferrets (25). Experimental infections of Syrian hamsters with HeV is lethal, and animals show disease similar to that of human cases, including respiratory and neurological symptoms, depending on the dose (11; unpublished data). In this model, viral RNA can be detected in various organs of infected hamsters, including brain, lung, kidney, heart, liver, and spleen. The main histopathological findings included parenchymal infection in various organs, including the brain, with vasculitis and syncytial multinucleated endothelial cells in many blood vessels (11). While this model is useful in studying pathogenesis, it is limited in the availability of reagents to do so.There are currently no vaccines or treatments licensed for human use. Several in vitro studies have shown that ribavirin is effective against both HeV and NiV infection (1, 2, 29). An open-label ribavirin treatment trial was run during an outbreak of NiV in Malaysia in 1998 and reported to reduce mortality by 36% (6). Of the seven recorded human HeV cases, three patients were treated with ribavirin, one of whom survived (22). In the most recent outbreak of HeV in Australia, three additional people received ribavirin treatment in combination with chloroquine after suspected exposure to HeV-contaminated secretions from infected horses. While all three individuals survived, infection was not confirmed, and therefore it remains unknown whether the treatment had any beneficiary effect (ProMed-mail no. 20090826.2998). In addition, two animal studies in hamsters showed that ribavirin treatment delays but does not prevent death from NiV or HeV infection (8, 10). Therefore, an animal model with greater relevance to humans and that recapitulates the disease processes seen in human cases of HeV is needed to get a better answer to whether ribavirin might be effective against henipavirus infections. In addition, the U.S. FDA implemented the “Animal Efficacy Rule,” which specifically applies to the development of therapeutic products when human efficacy studies are not possible or ethical, such as is often the case with highly virulent pathogens like HeV (24). Essentially, this rule allows for the evaluation of vaccines or therapeutics using data derived from studies carried out in at least two animal models. The licensure of any therapeutic modalities for HeV will require a thorough evaluation of HeV pathogenesis in nonhuman primates (NHPs).In the present study, we report the development and characterization of a new nonhuman primate (NHP) model of lethal HeV infection in the African green monkey (AGM). The pathogenesis and disease progression in the AGM upon HeV infection essentially mirrored the lethal disease episodes seen among human cases of HeV. Using this new model, the efficacy of ribavirin treatment against lethal challenge with HeV was examined. Here we have shown that ribavirin treatment can significantly delay but not prevent death of AGMs from lethal HeV infection. In addition to severe respiratory symptoms in all animals, prolonged disease progression in ribavirin-treated animals was also marked by the appearance of neurological symptoms.     

Early limited nitrosamine exposures exacerbate high fat diet-mediated type 2 diabetes and neurodegeneration

Background

Type 2 diabetes mellitus (T2DM) and several types of neurodegeneration, including Alzheimer's, are linked to insulin-resistance, and chronic high dietary fat intake causes T2DM with mild neurodegeneration. Intra-cerebral Streptozotocin, a nitrosamine-related compound, causes neurodegeneration, whereas peripheral treatment causes DM.

Hypothesis

Limited early exposures to nitrosamines that are widely present in the environment, enhance the deleterious effects of high fat intake in promoting T2DM and neurodegeneration.

Methods

Long Evans rat pups were treated with N-nitrosodiethylamine (NDEA) by i.p. injection, and upon weaning, they were fed with high fat (60%; HFD) or low fat (5%; LFD) chow for 8 weeks. Cerebella were harvested to assess gene expression, and insulin and insulin-like growth factor (IGF) deficiency and resistance in the context of neurodegeneration.

Results

HFD ± NDEA caused T2DM, neurodegeneration with impairments in brain insulin, insulin receptor, IGF-2 receptor, or insulin receptor substrate gene expression, and reduced expression of tau and choline acetyltransferase (ChAT), which are regulated by insulin and IGF-1. In addition, increased levels of 4-hydroxynonenal and nitrotyrosine were measured in cerebella of HFD ± NDEA treated rats, and overall, NDEA+HFD treatment reduced brain levels of Tau, phospho-GSK-3β (reflecting increased GSK-3β activity), glial fibrillary acidic protein, and ChAT to greater degrees than either treatment alone. Finally, pro-ceramide genes, examined because ceramides cause insulin resistance, oxidative stress, and neurodegeneration, were significantly up-regulated by HFD and/or NDEA exposure, but the highest levels were generally present in brains of HFD+NDEA treated rats.

Conclusions

Early limited exposure to nitrosamines exacerbates the adverse effects of later chronic high dietary fat intake in promoting T2DM and neurodegeneration. The mechanism involves increased generation of ceramides and probably other toxic lipids in brain.     

Sputum neutrophils as a biomarker in COPD: findings from the ECLIPSE study

Introduction

The percentage of neutrophils in sputum are increased in COPD patients, and may therefore be a biomarker of airway inflammation. We studied the relationships between sputum neutrophils and FEV₁, health status, exacerbation rates, systemic inflammation and emphysema, and long term variability at 1 year.

Methods

Sputum samples were obtained from 488 COPD patients within the ECLIPSE cohort. 359 samples were obtained at baseline, and 297 after 1 year. 168 subjects provided samples at both visits. Serum interleukin-6 (IL-6), IL-8, surfactant protein D and C-reactive protein levels were measured by immunoassays. Low-dose CT scans evaluated emphysema.

Results

Sputum neutrophil % increased with GOLD stage. There was a weak association between % sputum neutrophils and FEV₁ % predicted (univariate r² = 0.025 and 0.094 at baseline and year 1 respectively, p < 0.05 after multivariate regression). Similar weak but significant associations were observed between neutrophil % and health status measured using the St Georges Respiratory Questionairre. There were no associations between neutrophils and exacerbation rates or emphysema. Associations between sputum neutrophils and systemic biomarkers were non-significant or similarly weak. The mean change over 1 year in neutrophil % was an increase of 3.5%.

Conclusions

Sputum neutrophil measurements in COPD are associated weakly with FEV₁ % predicted and health status. Sputum neutrophil measurements were dissociated from exacerbation rates, emphysema and systemic inflammation.     

Exenatide does not evoke pancreatitis and attenuates chemically induced pancreatitis in normal and diabetic rodents

The risk of developing pancreatitis is elevated in type 2 diabetes and obesity. Cases of pancreatitis have been reported in type 2 diabetes patients treated with GLP-1 (GLP-1R) receptor agonists. To examine whether the GLP-1R agonist exenatide potentially induces or modulates pancreatitis, the effect of exenatide was evaluated in normal or diabetic rodents. Normal and diabetic rats received a single exenatide dose (0.072, 0.24, and 0.72 nmol/kg) or vehicle. Diabetic ob/ob or HF-STZ mice were infused with exenatide (1.2 and 7.2 nmol·kg(-1)·day(-1)) or vehicle for 4 wk. Post-exenatide treatment, pancreatitis was induced with caerulein (CRN) or sodium taurocholate (ST), and changes in plasma amylase and lipase were measured. In ob/ob mice, plasma cytokines (IL-1β, IL-2, IL-6, MCP-1, IFNγ, and TNFα) and pancreatitis-associated genes were assessed. Pancreata were weighed and examined histologically. Exenatide treatment alone did not modify plasma amylase or lipase in any models tested. Exenatide attenuated CRN-induced release of amylase and lipase in normal rats and ob/ob mice but did not modify the response to ST infusion. Plasma cytokines and pancreatic weight were unaffected by exenatide. Exenatide upregulated Reg3b but not Il6, Ccl2, Nfkb1, or Vamp8 expression. Histological analysis revealed that the highest doses of exenatide decreased CRN- or ST-induced acute inflammation, vacuolation, and acinar single cell necrosis in mice and rats, respectively. Ductal cell proliferation rates were low and similar across all groups of ob/ob mice. In conclusion, exenatide did not modify plasma amylase and lipase concentrations in rodents without pancreatitis and improved chemically induced pancreatitis in normal and diabetic rodents.     

Two amphibian diseases, chytridiomycosis and ranaviral disease, are now globally notifiable to the World Organization for Animal Health (OIE): an assessment

The global trade in amphibians entails the transport of tens of millions of live animals each year. In addition to the impact harvesting wild animals can have on amphibian populations, there is mounting evidence that the emerging pathogens Batrachochytrium dendrobatidis and ranaviruses, the aetiological agents of chytridiomycosis and ranaviral disease, respectively, are spread through this trade. The link between these pathogens and amphibian declines and extinctions suggests that the epidemiological impact of the trade is significant and may negatively affect conservation and trade economics. Here we present a brief assessment of the volume of the global trade in live amphibians, the risk of individuals harboring infection, and information on the recent listing by the World Organization for Animal Health (OIE) of chytridiomycosis and ranaviral disease in the OIE Aquatic Animal Health Code. This listing made chytridiomycosis and ranaviral disease internationally notifiable diseases and thus subject to OIE standards, which aim to assure the sanitary safety of international trade in live amphibians and their products.